von Willebrand factor (VWF) is a large multimeric plasma glycoprotein crucial in the maintenance of hemostasis by functioning as both an antihemophilic factor carrier and a platelet-vessel wall mediator in the blood coagulation system, mainly by mediating tethering and adhesion of circulating platelets at sites of vascular injury. Mutations in this gene or deficiencies in this protein result in von Willebrand's disease (VWD).
VWF is expressed by endothelial cells and megakaryocytes. It is synthesized as 250-kDa monomers, which undergo intracellular processing, glycosylation, multimerization and propeptide removal that leads to formation of mature VWF multimers.
VWF multimeric size is modulated by the plasma metallopeptidase ADAMTS13 (a disintegrin and metallopeptidase with thrombospondin type I motif, member 13, a “cleaving protease”), which cleaves at a single site in the VWF A2 domain (AA1498-1665; UniProtKB/Swiss-Pro database; Accession: P04275. SEQ ID NO:2) between Y1605 and M1606.
ADAMTS13 is a protease that is activated in the presence of barium and other metal ions. ADAMTS13 has been demonstrated to degrade full-length multimeric vWF into multimers of smaller size and into lower molecular weight polypeptides or peptides. For this reason, the ADAMTS13 protease has been termed vWF-cleaving protease or the “ATS protease”. The activity of the protease has been demonstrated to be reduced in patients with Thrombotic Thrombocytopenia Purpura (TTP).
Severe deficiency of the protease has been described in patients with chronic relapsing TTP, a deficiency that may be inherited or acquired as a result of an autoimmune mechanism.
In the past, assays for the presence or absence of ADAMTS13 utilized a cumbersome technique in which plasma from a patient is incubated with exogenous multimeric vWF in the presence of barium chloride on the surface of a membrane floating on a buffer containing 1.5 molar urea. More recently an alternative method has been developed by Kokame et al. (Kokame, K., Y. Nobe, Y. Kokubo, A. Okayama, and T. Miyata. 2005. FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay. Br. J. Haematol. 129:93-100. See also Wu J J, Fujikawa K, McMullen B A, Chung D W. Characterization of a core binding site for ADAMTS13 in the A2 domain of von Willebrand factor. Proc Natl Acad Sci USA. 2006; 103: 18470-4.). Kokame's method utilizes a polypeptide substrate for ADAMTS13 activity, wherein the substrate is 73 amino acid residues in the A2 domain of VWF, called VWF73. FRETS-VWF73 is within this domain and the 73-amino-acid polypeptide sequence corresponds to the region from D1596 to R1668 of VWF (see SEQ ID NO:6 herein), Q1599 and N1610 when substituted with A2pr(Nma) and A2pr(Dnp) respectively. Several assays have been developed using SEQ ID NO:6. VWF73-based ADAMTS13 assays have the potential to contribute to improved clinical treatments.
However, the de novo synthesis of SEQ ID NO:6 is difficult and the FRETS-VWF73 substrate works near the UV spectrum. The signal that is generated therefore suffers from heavy contribution of autofluorescence which can be exacerbated by the fact that the assay is homogeneous, i.e. is performed in a single step without washing away the plasma, one of the major contributors to the autofluorescence noise. Because of its susceptibility to autofluorescence, an assay based on the FRETS-VWF73 substrate is very sensitive to dust microparticles, potentially resulting in poor replicates and aberrant results. Furthermore, FRETS-VWF73 substrate assays typically result in a non-linear calibration curve which can result in low accuracy below 10% of ADAMTS13 activity. This is problematic since the resolution of ADAMTS13 activity at between 0-10% is important to clinicians to confirm the diagnosis of TTP and to monitor and fine tune the therapeutic intervention (such as plasma exchange). Further, ADAMTS13 activity assays using a SEQ ID NO:6 polypeptide suffer from poor sensitivity.
As a result, there is a need in the art for an improved ADAMTS13 polypeptide substrate. The present invention seeks to address this need.